Peptidoglycan Biogenesis in Escherichia Coli

总结 细菌细胞生物学的一个主要目标是了解细菌的组装和生长的机制。 手机信封除了解决一个基本的生物学问题外，这一领域的研究还 对人类健康的重大影响。包膜既是抗生素的主要靶点， 革兰氏阴性菌的情况下，一个强大的障碍，阻止药物到达他们的目标。因此，在本发明中， 了解构建革兰氏阴性包膜所需的机制将有助于确定 在抗生素开发过程中出现了新的弱点。肽聚糖（PG）细胞壁层 包封对于电池形状和完整性是至关重要的。它是由交联连接的长聚糖组成的 形成网状结构，包围并保护细胞质膜 从渗透溶解中分离出来。在大肠大肠杆菌和许多其他杆菌，细胞伸长和细胞分裂的过程是 由多蛋白质细胞壁合成机器分别称为杆系统和divisome进行。 杆系统由肌动蛋白样蛋白MreB的细丝组织，而微管蛋白样蛋白FtsZ 控制着细胞分裂尽管经过多年的研究，这些机器中蛋白质的功能仍然存在 令人惊讶的不明确。直到最近，人们甚至还不清楚哪些酶在这些酶中合成PG。 配合物因为它们是已知具有PG聚糖聚合酶活性的唯一因子，所以A类 青霉素结合蛋白（aPBPs）传统上被认为填补了这一作用。然而，我们改变了这一点， 通过证明杆系统中的SEDS（形状、伸长、分裂和孢子形成）蛋白质来观察 （RodA）和分裂体（FtsW）具有PG聚合酶活性，并与PG交联一起起作用 称为B类PBP（bPBP）的酶来构建细胞壁。因此，我们的研究结果使我们提出了一个 细胞壁合成的新模型，其中SEDS-bPBP复合物形成细胞壁的核心PG激酶 有组织的机器，其中RodA-PBP 2和FtsW-PBP 3包括杆系统和分裂酶， 分别本提案中描述的实验将建立在我们最近的突破基础上， 一个新开发的遗传系统的优势，用于分离编码失活或过度活跃的突变体 杆系统。分离的几种突变体为确定RodA聚合酶活性如何影响细胞的生长提供了基础。 在杆系统内调节并与PBP 2的交联活性偶联。另外的遗传 还将启动旨在确定其他保守但特征不明确的功能的分析 Rod系统的组分及其在调节核心RodA-PBP 2活性中的潜在作用 合成酶最后，将启动生物化学和遗传学研究，以了解相关的FtsW-PBP 3 合酶在分裂体内受到调节。总的来说，研究结果将大大促进我们对 细胞壁生物合成的多蛋白质PG合成机制，所获得的知识将有助于发现 针对这些系统的新型抗菌剂。

SUMMARY A major goal of bacterial cell biology is to understand the mechanisms underlying the assembly and growth of the cell envelope. In addition to addressing a fundamental biological question, studies in this area have significant consequences for human health. The envelope serves as both a major target for antibiotics and, in the case of gram-negative bacteria, a formidable barrier that prevents drugs from reaching their target. Thus, understanding of the mechanisms required for construction of the gram-negative envelope will help identify new vulnerabilities in the process to target for antibiotic development. The peptidoglycan (PG) cell wall layer of the envelope is critical for cell shape and integrity. It is composed of long glycans connected by crosslinks between attached peptides to form a net-like structure that surrounds and protects the cytoplasmic membrane from osmotic lysis. In E. coli and many other bacilli, the processes of cell elongation and cell division are carried out by multi-protein cell wall synthetic machines called the Rod system and the divisome, respectively. The Rod system is organized by filaments of the actin-like protein MreB whereas the tubulin-like protein FtsZ governs cell division. Despite years of study, the function of proteins within these machineries have remained surprisingly ill-defined. Until recently, it has even been unclear which enzymes synthesize PG within these complexes. Because they were the only factors known to possess PG glycan polymerase activity, the class A penicillin-binding proteins (aPBPs) have traditionally been thought to fill this role. However, we changed this view by demonstrating that SEDS (shape, elongation, division, and sporulation) proteins in the Rod system (RodA) and divisome (FtsW) have PG polymerase activity and work in conjunction with PG crosslinking enzymes called class B PBPs (bPBPs) to build the cell wall. Our findings have therefore led us to propose a new model for cell wall synthesis where SEDS-bPBP complexes form the core PG synthases of cytoskeletally organized machineries, with RodA-PBP2 and FtsW-PBP3 comprising the Rod system and divisome synthases, respectively. The experiments described in this proposal will build on our recent breakthrough by taking advantage of a newly developed genetic system for the isolation of mutants encoding inactive or hyperactive Rod systems. Several mutants isolated provide a foundation for defining how RodA polymerase activity is regulated within the Rod system and coupled with the crosslinking activity of PBP2. Additional genetic analyses will also be initiated aimed at defining the function of other conserved yet poorly characterized components of the Rod system and their potential role in regulating the activity of the core RodA-PBP2 synthase. Finally, biochemical and genetic studies will be initiated to understand how the related FtsW-PBP3 synthase is regulated within the divisome. Overall, the results will significantly advance our understanding of cell wall biogenesis by multi-protein PG synthetic machineries, and the knowledge gained will aid the discovery of new classes of antibacterial agents that target these systems.